The present invention relates to detecting and isolating phosphorylated molecules using phosphoaffinity materials containing one or more hydrated metal oxides, such as yttrium oxide, yttrium aluminum garnet and titanium dioxide.
Cells of the body contain many types of molecules that vary in function, size, lifetime, and numerous other characteristics. Some of these molecules are unchanged during their lifetime within the body, while other molecules become modified through chemical reactions. The modifications can be indicative of particular cell states, including normal states as well as abnormal states caused by injury, infection and disease.
Proteins, for example, are often chemically modified during their lifetime in the body. A protein can be modified during and after its synthesis, or both, and the modification can change the size and the structure of the protein, which in turn can result in changing the protein's function or behavior in the cell. An example of a modification of a protein is the addition of a phosphate group (phosphorylation).
Reversible phosphorylation of threonine, serine, and tyrosine residues on proteins by enzymes called kinases (which add a phosphate) and phosphatases (which remove the phosphate) plays an important role in regulating many cell processes, such as growth and cell cycle control. Phosphorylation can occur sequentially from one protein to another, resulting in a series of activations called a “phosphorylation cascade,” which is a type of “signal transduction pathway.” Phosphorylation cascades are recognized as signaling networks that direct growth, death, and differentiation of cells—the critical signals for maintaining normal cells in the body. At any given moment in a cell, determination of phosphorylation states of proteins can indicate a signal transduction state, for example an “on” or “off” state of cell growth.
Many cellular processes are regulated by reversible phosphorylation of proteins and upwards of 30% of the total complement of proteins expressed by human cells are likely to be phosphorylated at some point during their existence. Determination of protein phosphorylation state is thus important for identifying protein kinase substrates, as well as revealing the on/off state of signal transduction pathways. The on/off state of signal transduction pathways can be important to understanding pathophysiological processes, such as cancer. To better understand such signal transduction pathways, efforts are underway within the research community to identify phosphorylated proteins of various cell types under different cellular conditions, such as normal and diseased conditions. Determination of differences in phosphorylation that occur under normal and diseased conditions can be used, for example, in development of diagnostic and other medical tests.
Given the important role of phosphorylation in signal transduction pathways, analysis of phosphorylation events that occur within the entire complement of proteins expressed by cells (phosphoproteome analysis), is useful for understanding a range of cellular processes. Phosphoproteome analysis likely will reveal insight into complex biological processes, such as differentiation, growth control and regulated cell death. Accordingly, phosphoproteome analysis is expected to contribute to development of diagnostic and prognostic tests, improve aspects of clinical trials, and provide indications of drug safety and efficacy during drug development.
One challenge in the field of phosphoproteome analysis is developing accurate methods for global evaluation of protein phosphorylation levels. Global analysis of protein phosphorylation is an analytical challenge because signaling phosphoproteins are typically present in low abundance within cells. Analytical methods that improve global analysis of protein phosphorylation can contribute to development of medical tests, such as tests that can simultaneously test for multiple phosphoprotein biomarkers. This type of test is expected to be helpful for detecting diseases and conditions for which single diagnostic markers are unfeasible or unavailable.
Thus, the ability to detect and/or isolate phosphoproteins is useful for cellular research as well as medical test development, given the central role of phosphorylation in many disease processes. Improved approaches for phosphomolecule isolation and detection would accelerate protein phosphorylation global analysis and related general and biomedical phosphomolecule research.